Brefeldin A (BFA) is a fungal metabolite with a broad range of biological activities, including the anti-fugal and anti-tumor activities. In the past few years there has been tremendous interest in its effect on cells, since it has been found to have the unique ability to interfere with vesicular trafficking. Among its effects, it has the ability to disperse the Golgi complex, returning its components to the endoplasmic reticulum (ER), and prevent newly synthesized proteins destined for the cell surface from leaving the ER. To better understand the mechanism of BFA action we conjugated it to two fluorescent dyes and studied its intracellular localization. Both conjugates maintained their biological activity, and both specifically localized to the ER in live or aldehyde-fixed cells. The interaction with the ER-membrane is probably due to interaction with lipids, and not with ER proteins. These conjugates are the first dyes that bind the ER without binding other prominent membrane bound compartments, and should prove useful as probes for ER structure in living cells under various conditions. Their ability to stain the ER of fixed cells will be of great use as markers of the ER in cytochemical studies. Finally, the interaction of BFA with the ER might be critical to its biological activity.